The basic contention that the determinant of primary male sex in mammals is the molecule (or molecules) bearing H-Y antigen is now well-substantiated. The corollary that testicular differentiation should invariably be associated with presence of H-Y antigen, and ovarian differentiation with absence of H-Y antigen, regardless of karyotype or secondary sex phenotype, has withstood repeated and extensive testing under stringent conditions and in a wide variety of biological circumstances. Current research is moving toward substantiation of further theory: (i) that testicular organogenesis depends on attachment of disseminated H-Y to a gonad-specific receptor, and that defectiveness or absence of this receptor entails ovarian organogenesis in XY gonads, and (ii) that this receptor is present on uncommitted gonads of XX as well as XY embryos, hence development of testes or ovotestes in XX primordia abnormally exposed to H-Y. Technical advances (projected in this grant proposal) are likely to be critical in this work, especially radiolabeling of H-Y (by feeding radioactive amino acids to cultured Daudi or Sertoli cells, which release free H-Y), and immunoprecipitation of labeled H-Y with H-Y antibody (from conventional immunization, multiparous mice, or hybridoma clones now being screened for precipitating H-Y antibody). Application of these methods should facilitate the proposed studies concerning (i) the chemical structure of H-Y, (ii) the relative roles of Y-linked and X-linked genes in H-Y synthesis, (iii) aberrant gonadal development, as in the freemartin, and (iv) agents that may promote ovarian differentiation by impeding or forestalling reaction of H-Y and its receptor. It is further proposed to extend our studies of the occurrence and actions of H-Y in primitive vertebrates such as the clawed frog, Xenopus laevis, because organisms in which rigid sex chromosomal and sex phenotypic dimorphism are lacking, and in which sex can be experimentally manipulated, e.g. by exposure to steroid hormones, offer models that may elucidate evolution of primary sex determination in the higher forms.